The C-terminal SMMHC region of the inv(16) fusion protein contains a repression domain. (A) Schematic diagrams of internal deletion mutants of inv(16). (B) NIH 3T3 cells were transfected with 50 ng of plasmids expressing inv(16) or inv(16) mutants and 1 μg of thymidine kinase-firefly luciferase reporter plasmid. Fold repression was calculated after correcting for transfection efficiency using a plasmid expressing SEAP. Levels of repression are the averages ± standard errors of three experiments. Empty expression vector was used as a control. Note that error was too small to graph in a few samples. (C) Immunoblot analysis of internal deletion mutants of inv(16) expressed in Cos7 cells (100 μg of protein from whole-cell lysate was loaded for each mutant).

The C-terminal 163 aa of the inv(16) fusion protein are sufficient for repression. (A) Schematic diagrams of inv(16) C-terminal fragments linked to the GAL4 DNA-binding domain. (B) NIH 3T3 cells were transfected with 50 ng of plasmids expressing inv(16) or the indicated mutants and 1 μg of thymidine kinase-firefly luciferase reporter plasmid. Fold repression was calculated after correcting for transfection efficiency using a plasmid expressing SEAP. Levels of repression are the averages ± standard errors of three experiments. Empty expression vector was used as a control. Note that error was too small to graph in a few samples. (C) Immunoblot analysis of internal deletion mutants of inv(16) expressed in Cos7 cells (100 μg of protein from whole-cell lysate was loaded for each mutant).

The inv(16) fusion protein interacts with mSin3A. (A) Cos7 cells were transfected with pCMV5-GAL4-inv. Cell lysates were prepared in PBS containing 0.5% Triton X-100, 0.1% DOC, and 0.1% SDS. Endogenous mSin3A was precipitated with anti-Sin3A (αmSin3A) polyclonal antibody. Copurifying GAL4-inv(16) was detected by immunoblotting with antibodies to the GAL4 DNA-binding domain. Lanes: WCL, whole-cell lysate; control, lysate incubated with protein A-agarose (no primary immunoglobulin G [IgG]); αmSin3A, lysate incubated with mSin3A IgG; αmSin3A+pep, lysate incubated with peptide-blocked mSin3A IgG. (B) ME-1 cells were lysed in PBS containing 0.5% Triton X-100, 0.3% DOC, and 0.2% SDS. Endogenous mSin3A and HDAC2 were precipitated with anti-mSin3A (αmSin3A) and anti-HDAC2 (αHDAC2) polyclonal antibodies. Copurifying inv(16) was detected by immunoblotting with anti-CBFβ. The position of a nonspecific (NS) band is indicated by the arrow.

The C-terminal domain of CBFβ/SMMHC is sufficient for association with mSin3A. (A) Cos7 cells were transfected with GAL4-inv(16) and the GAL4-inv(16) internal deletion mutants (Fig. 1A). Cell lysates were prepared in PBS containing 0.5% Triton X-100, 0.1% DOC, and 0.1% SDS. Endogenous mSin3A was precipitated with anti-mSin3A antibody. Copurifying proteins were analyzed by immunoblot analysis using antibodies to mSin3A and the GAL4 DNA-binding domain. The lysates were incubated with mSin3A immunoglobulin G (IgG) (IP lanes) or with peptide-blocked mSin3A IgG (C lanes). (B) Same as panel A except that lysates were prepared in PBS containing 0.5% Triton X-100. Nonspecific bands are indicated by the asterisks. W, whole-cell lysate. (C) Cos7 cells were transfected with GAL4-inv(16)449-611 or GAL4-inv(16)532-611 (Fig. 2A). Cells were lysed in PBS with 0.5% Triton X-100 and 0.1% DOC followed by mSin3A immunoprecipitation. Immunoblot analysis was performed as in panel A.

The inv(16) fusion protein interacts with HDAC8. (A) Cos7 cells were transfected with pCMV5-GAL4-inv(16) and FLAG-tagged HDAC1 to HDAC6 and HDAC9 (HDRP fragment), HA-tagged HDAC7, or Myc-tagged HDAC8. Cell lysates were prepared in PBS containing 0.5% Triton X-100. Co-IPs were performed using antibodies to the tagged HDACs and were analyzed by immunoblotting with antibodies to FLAG (αFlag), HA (αHA), Myc (αMyc), and the GAL4 DNA-binding domain (αGAL). Cells transfected with GAL4-inv(16) alone are shown in c lanes. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blots. (B) GAL4-inv(16) or GAL4-inv(16)Δ2-11 expressing plasmids were cotransfected with (+) Myc-tagged HDAC8 and analyzed as in panel A. WCL, whole-cell lysate.

AML-1 binds HDAC1, HDAC3, and HDAC9. AML-1 was coexpressed with HDAC1 to -8 or -9 (HDRP fragment) in Cos7 cells and coimmunoprecipitated with anti-FLAG, Myc, or HA in PBS containing 0.5% Triton X-100, 0.1% DOC, and 0.1% SDS (top blot). Copurifying AML-1 was detected by immunoblot analysis using anti-RHD (Calbiochem). The bottom blot shows HDAC and AML-1B expression in whole-cell lysate. The positions of the HDACs (shown in the figure without the HDAC prefix) are indicated by the arrows. α-AML1 and α-Rhd, anti-AML1 antibodies. Lane C, AML1 expressed in the absence of HDACs.

Mapping the HDAC8 binding domain in CBFβ/SMMHC. (A) Cos7 cells were cotransfected with Myc-tagged HDAC8 and GAL4-inv(16) or one of the GAL4-inv(16) deletion mutants (Fig. 1A). Cells were lysed in PBS containing 0.5% Triton X-100. HDAC8 was immunoprecipitated with antibodies to the Myc tag (αMyc), and copurifying proteins were analyzed with antibodies to the GAL4 DNA-binding domain and the Myc epitope tag (αGAL/αMyc). Lanes: W, whole-cell lysate; IP, lysates immunoprecipitated with anti-Myc; C, Cos7 cells transfected with only GAL4-inv(16) or the GAL4-inv(16) deletion mutants and lysates immunoprecipitated with anti-Myc. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the blot. The positions of nonspecific bands are indicated by asterisks. (B) Cos7 cells were cotransfected with Myc-HDAC8 and GAL4-inv(16) or one of the GAL4-inv(16) deletion mutants (Fig. 2A) and analyzed as in panel A except that PBS contained 0.5% Triton X-100 and 0.3% DOC.

ACD is required for transcriptional repression and interaction with corepressors. (A) Schematic of inv(16)ΔACD mutant. (B) Cos7 cells were transfected with GAL4-inv(16) or GAL4-inv(16)ΔACD. Cells were fractionated in isotonic buffer containing 0.5% NP-40 by low-speed centrifugation. Equal proportions of the cytoplasmic (C) and nuclear (N) fractions were analyzed by SDS-PAGE and immunoblotting with antibodies to phospholipase Cγ1 (αPLCγ1), GAL4 DNA-binding domain (αGAL4DBD), and HDAC2 (αHDAC2). Untransfected Cos7 cells were used as a control. (C) NIH 3T3 cells were transfected with increasing amounts of GAL4-inv(16)- or GAL4-inv(16)ΔACD-expressing plasmid, 1 μg of thymidine kinase-firefly luciferase reporter plasmid, and 200 ng of pCMV-SEAP. Fold repression was calculated after correcting for transfection efficiency using SEAP; each value is the average ± standard error of two experiments. Empty expression vector was used as a control. Note that error was too small to graph in several samples. (D) Cos7 cells were transfected with pCMV5-GAL4ΔACD. Cell lysates were prepared in PBS containing 0.5% Triton X-100. Co-IPs were performed using anti-mSin3A (αmSin3A) polyclonal antibody and were analyzed by immunoblotting with antibodies to mSin3A and the GAL4 DNA-binding domain. WCL, whole-cell lysate; control, immunoprecipitation using peptide-blocked anti-mSin3A. (E) Cos7 cells were transfected with pCMV5-GAL4inv(16) or pCMV5-GAL4ΔACD alone or with Myc-tagged HDAC8. Cell lysates were prepared in PBS containing 0.5% Triton X-100. Co-IPs were performed using anti-Myc and were analyzed by immunoblotting with antibodies to Myc and the GAL4 DNA-binding domain.

Artificial multimerization of the inv(16) fusion protein does not induce corepressor binding. (A) Schematic diagrams of mutants of inv(16) and the p53 tetramerization domain. (B) Cos7 cells were transfected with pCMV5-GAL4-p53tet, pCMV5-GAL4-inv(16) or plasmids expressing mutant inv(16) proteins. Cell lysates were prepared in PBS containing 0.5% Triton X-100. Co-IPs were performed using anti-mSin3A (αmSin3A) and were analyzed by immunoblotting with antibodies to mSin3A and the GAL4 DNA-binding domain (αGAL4DBD). Lysates were incubated with mSin3A immunoglobulin G (IgG) (IP lanes) or with peptide-blocked mSin3A IgG (C lanes). (C) NIH 3T3 cells were transfected with 100 ng of plasmids expressing inv(16)- or inv(16) mutants or p53tet and 1 μg of thymidine kinase-firefly luciferase reporter plasmid. Fold repression was calculated after correcting for transfection efficiency using a plasmid expressing SEAP. Levels of repression are the averages ± standard errors of three experiments. Empty expression vector was used as a control. Note that error was too small to graph in some samples.

TSA treatment impairs inv(16)-mediated transcriptional repression. (A) inv(16)-expressing plasmid (100 ng) was cotransfected with 1 μg of thymidine kinase (TK)-firefly luciferase reporter plasmid and 100 ng of pRL-TK. After transfection, TSA at the indicated concentrations was added to the culture medium for 48 h. Fold repression was calculated after normalizing to pRL-TK activity. Note that TSA affected the control (empty expression vector) to some degree (data not shown), and the values reported are relative to controls that were untreated (bar 0) or treated with TSA (bars 10, 25, and 50). Levels of repression are the averages ± standard errors of three experiments. Note that error was too small to graph in one sample. (B) Immunoblot analysis of transfected NIH 3T3 cell lysates from the experiment in panel A.